Tn7 is an E. coli transposable element that encodes resistance to trimethoprim, spectinomycin and streptomycin. In contrast to most transposable elements, Tn7 transposes with high efficiency to a specific site called attTn7. Tn7 also transposes with reduced specificity and efficiency to secondary (2 degrees) sites. This work will define the components of Tn7 transposition and examine the mechanism of this novel transposition reaction. The basic strategy to define the components will be to examine the transposition properties of variants of the components. The target and donor sites will be analyzed by defining the nucleotide sequences that promote the activity of attTn7 and the ends of Tn7. The critical features of target sites will also be analyzed by examining the structure of 2 degrees sites including one preferred site in Caulobacter crescentus. The donor site will also be analyzed by comparing the activities of the left and right ends of Tn7 and by determining whether the sequences that flank these ends in the donor site participate directly in transposition. The genetic organization of Tn7 will be analyzed by identifying Tn7-encoded functions that promote transposition. The roles of these functions will be explored by determining what steps in transposition they mediate. The mechanism of transposition will also be analyzed by examining the formation of cointegrates and by looking directly at DNA breakage and joining reactions at the target and donor sites. Elucidation of the mechanism of Tn7 transposition will contribute to the understanding of the rapid dispersal of antibiotic resistance. It will also provide insights into the mechanisms of other DNA rearrangements that underlie a wide variety of important cellular processes in both procaryotes and eucaryotes. Comparison of the basis of site-specificity in this replicative recombination with the basis of site-specificity in conservative recombination should be particularly interesting.